Modified protein kinase A-specific oligonucleotides and methods of their use

ABSTRACT

Disclosed are synthetic, modified oligonucleotides complementary to, and capable of down-regulating the expression of, nucleic acid encoding protein kinase A subunit RI α . The modified oligonucleotides have from about 15 to about 30 nucleotides and are hybrid, inverted hybrid, or inverted chimeric oligonucleotides. Also disclosed are therapeutic compositions containing such oligonucleotides and methods of using the same.

This application claims the benefit of U.S. Ser. No. 60/040,740, filedon Mar. 12, 1997, and is a continuation-in-part of U.S. Ser. No.08/532,979, filed Sep. 22, 1995, (now U.S. Pat. No. 5,969,117), which isa continuation-in-part of U.S. Ser. No. 08/516,454, filed Aug. 17, 1995(now U.S. Pat. No. 5,652,356).

FIELD OF THE INVENTION

The present invention relates to cancer therapy. More specifically, thepresent invention relates to the inhibition of the proliferation ofcancer cells using modified antisense oligonucleotides complementary tonucleic acid encoding the protein kinase A RI_(α) subunit.

BACKGROUND OF THE INVENTION

The development of effective cancer therapies has been a major focus ofbiomedical research. Surgical procedures have been developed and used totreat patients whose tumors are confined to particular anatomical sites.However, at present, only about 25% of patients have tumors that aretruly confined and amenable to surgical treatment alone (Slapak et al.in Harrison's Principles of Internal Medicine (Isselbacher et al., eds.)McGraw-Hill, Inc., NY (1994) pp. 1826-1850). Radiation therapy, likesurgery, is a local modality whose usefulness in the treatment of cancerdepends to a large extent on the inherent radiosensitivity of the tumorand its adjacent normal tissues. However, radiation therapy isassociated with both acute toxicity and long term sequelae. Furthermore,radiation therapy is known to be mutagenic, carcinogenic, andteratogenic (Slapak et al., ibid.).

Systemic chemotherapy alone or in combination with surgery and/orradiation therapy is currently the primary treatment available fordisseminated malignancies. However, conventional chemotherapeutic agentswhich either block enzymatic pathways or randomly interact with DNAirrespective of the cell phenotype, lack specificity for killingneoplastic cells. Thus, systemic toxicity often results from standardcytotoxic chemotherapy. More recently, the development of agents thatblock replication, transcription, or translation in transformed cells,and at the same time defeat the ability of cells to become resistant,has been the goal of many approaches to chemotherapy.

One strategy is to down regulate the expression of a gene associatedwith the neoplastic phenotype in a cell. A technique for turning off asingle activated gene is the use of antisense oligodeoxynucleotides andtheir analogues for inhibition of gene expression (Zamecnik et al.(1978) Proc. Natl. Acad. Sci. (USA) 75:280-284). An antisenseoligonucleotide targeted at a gene involved in the neoplastic cellgrowth should specifically interfere only with the expression of thatgene, resulting in arrest of cancer cell growth. The ability tospecifically block or down-regulate expression of such genes provides apowerful tool to explore the molecular basis of normal growthregulation, as well as the opportunity for therapeutic intervention(see, e.g., Cho-Chung (1993) Curr. Opin. Thera. Patents 3:1737-1750).The identification of genes that confer a growth advantage to neoplasticcells as well as other genes causally related to cancer and theunderstanding of the genetic mechanism(s) responsible for theiractivation makes the antisense approach to cancer treatment possible.

One such gene encodes the RI_(α) subunit of cyclic AMP (cAMP)-dependentprotein kinase A (PKA) (Krebs (1972) Curr. Topics Cell. Regul.5:99-133). Protein kinase is bound by cAMP, which is thought to have arole in the control of cell proliferation and differentiation (see,e.g., Cho-Chung (1980) J. Cyclic Nucleotide Res. 6:163-167). There aretwo types of PKA, type I (PKA-I) and type II (PKA-II), both of whichshare a common C subunit but each containing distinct R subunits, RI andRII, respectively (Beebe et al. in The Enzymes: Control byPhosphorylation, 17(A):43-111 (Academic, New York, 1986). The R subunitisoforms differ in tissue distribution (Øyen et al. (1988) FEBS Lett.229:391-394; Clegg et al. (1988) Proc. Natl. Acad. Sci. (USA)85:3703-3707) and in biochemical properties (Beebe et al. in TheEnzymes: Control by Phosphorylation, 17(A):43-111 (Academic Press, NY,1986); Cadd et al. (1990) J. Biol. Chem. 265:19502-19506). The twogeneral isoforms of the R subunit also differ in their subcellularlocalization: RI is found throughout the cytoplasm; whereas RIIlocalizes to nuclei, nucleoli, Golgi apparatus and themicrotubule-organizing center (see, e.g., Lohmann in Advances in CyclicNucleotide and Protein Phosphorylation Research, 18:63-117 (Raven, NewYork, 1984; and Nigg et al. (1985) Cell 41:1039-1051).

An increase in the level of RI_(α) expression has been demonstrated inhuman cancer cell lines and in primary tumors, as compared with normalcounterparts, in cells after transformation with the Ki-ras oncogene ortransforming growth factor-α, and upon stimulation of cell growth withgranulocyte-macrophage colony-stimulating factor (GM-CSF) or phorbolesters (Lohmann in Advances in Cyclic Nucleotide and ProteinPhosphorylation Research, 18:63-117 (Raven, New York, 1984); andCho-Chung (1990) Cancer Res. 50:7093-7100). Conversely, a decrease inthe expression of RI_(α) has been correlated with growth inhibitioninduced by site-selective cAMP analogs in a broad spectrum of humancancer cell lines (Cho-Chung (1990) Cancer Res. 50:7093-7100). It hasalso been determined that the expression of RI/PKA-I and RII/PKA-II hasan inverse relationship during ontogenic development and celldifferentiation (Lohmann in Advances in Cyclic Nucleotide and ProteinPhosphorylation Research, Vol. 18, 63-117 (Raven, New York, 1984);Cho-Chung (1990) Cancer Res. 50:7093-7100). The RI_(α) subunit of PKAhas thus been hypothesized to be an ontogenic growth-inducing proteinwhose constitutive expression disrupts normal ontogenic processes,resulting in a pathogenic outgrowth, such as malignancy (Nesterova etal. (1995) Nature Medicine 1:528-533).

Antisense oligonucleotides directed to the RI_(α) gene have beenprepared. U.S. Pat. No. 5,271,941 describes phosphodiester-linkedantisense oligonucleotides complementary to a region of the first 100N-terminal amino acids of RI_(α) which inhibit the expression of RI_(α)in leukemia cells in vitro. In addition, antisense phosphorothioateoligodeoxynucleotides corresponding to the N-terminal 8-13 codons of theRI_(α) gene was found to reduce in vivo tumor growth in nude mice(Nesterova et al. (1995) Nature Med. 1:528-533).

Unfortunately, problems have been encountered with the use ofphosphodiester-linked (PO) oligonucleotides and somephosphorothioate-linked (PS) oligonucleotides. It is known thatnucleases in the serum readily degrade PO oligonucleotides. Replacementof the phosphodiester internucleotide linkages with phosphorothioateinternucleotide linkages has been shown to stabilize oligonucleotides incells, cell extracts, serum, and other nuclease-containing solutions(see, e.g., Bacon et al. (1990) Biochem. Biophys. Meth. 20:259) as wellas in vivo (Iversen (1993) Antisense Research and Application (Crooke,ed) CRC Press, 461). However, some PS oligonucleotides have been foundto exhibit an immunostimulatory response, which in certain cases may beundesirable. For example, Galbraith et al. (Antisense Res. & Dev. (1994)4:201-206) disclose complement activation by some PS oligonucleotides.Henry et al. (Pharm. Res. (1994) 11: PPDM8082) disclose that some PSoligonucleotides may potentially interfere with blood clotting.

There is, therefore, a need for modified oligonucleotides directed tocancer-related genes that retain gene expression inhibition propertieswhile producing fewer side effects than conventional oligonucleotides.

SUMMARY OF THE INVENTION

The present invention relates to modified oligonucleotides useful forstudies of gene expression and for the antisense therapeutic approach.The invention provides modified oligonucleotides that down-regulate theexpression of the RI_(α) gene while producing fewer side effects thanconventional oligonucleotides. In particular, the invention providesmodified oligonucleotides that demonstrate reduced mitogenicity, reducedactivation of complement and reduced antithrombotic properties, relativeto conventional oligonucleotides.

It is also known that some PS oligonucleotides cause animmunostimulatory response in subjects to whom they have beenadministered, which may be undesirable in some cases.

It is known that exclusively phosphodiester- or exclusivelyphosphorothioate-linked oligonucleotides directed to the first 100nucleotides of the RI_(α) nucleic acid inhibit cell proliferation.

It has now been discovered that modified oligonucleotides complementaryto the protein kinase A RI_(α) subunit gene inhibit the growth of tumorsin vivo. With at least the activity of a comparable PO- or PS-linkedoligonucleotide with fewer side effects.

This finding has been exploited to produce the present invention, whichin a first aspect, includes synthetic hybrid, inverted hybrid, andinverted chimeric oligonucleotides and compositions of matter forspecifically down-regulating protein kinase A subunit RI_(α) geneexpression with reduced side effects. Such inhibition of gene expressionis useful as an alternative to mutant analysis for determining thebiological function and role of protein kinase A-related genes in cellproliferation and tumor growth. Such inhibition of RI_(α) geneexpression can also be used to therapeutically treat diseases anddisorders that are caused by the over-expression or inappropriateexpression of the gene.

As used herein, the term “synthetic oligonucleotide” includes chemicallysynthesized polymers of three up to 50, preferably from about 15 toabout 30, and most preferably, 18 ribonucleotide and/ordeoxyribonucleotide monomers connected together or linked by at leastone, and preferably more than one, 5′ to 3′ internucleotide linkage.

For purposes of the invention, the term “oligonucleotide sequence thatis complementary to a genomic region or an RNA molecule transcribedtherefrom” is intended to mean an oligonucleotide that binds to thenucleic acid sequence under physiological conditions, e.g., byWatson-Crick base pairing (interaction between oligonucleotide andsingle-stranded nucleic acid) or by Hoogsteen base pairing (interactionbetween oligonucleotide and double-stranded nucleic acid) or by anyother means including in the case of a oligonucleotide binding to RNA,causing pseudoknot formation. Binding by Watson-Crick or Hoogsteen basepairing under physiological conditions is measured as a practical matterby observing interference with the function of the nucleic acidsequence.

In one preferred embodiment according to this aspect of the invention,the oligonucleotide is a core region hybrid oligonucleotide comprising aregion of at least two deoxyribonucleotides, flanked by 5′ and 3′ribonucleotide regions, each having at least four ribonucleotides. Ahybrid oligonucleotide having the sequence set forth in the SequenceListing as SEQ ID NO:4 is one particular embodiment. In someembodiments, each of the 3′ and 5′ flanking ribonucleotide regions of anoligonucleotide of the invention comprises at least four contiguous,2′-O-substituted ribonucleotides.

For purposes of the invention, the term “2′-O-substituted” meanssubstitution of the 2′ position of the pentose moiety with an -O-loweralkyl group containing 1-6 saturated or unsaturated carbon atoms, orwith an -O-aryl or allyl group having 2-6 carbon atoms, wherein suchalkyl, aryl or allyl group may be unsubstituted or may be substituted,e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy,alkoxy, carboxyl, carbalkoxyl, or amino groups; or with a hydroxy, anamino or a halo group, but not with a 2′-H group.

In some embodiments, each of the 3′ and 5′ flanking ribonucleotideregions of an oligonucleotide of the invention comprises at least one2′-O-alkyl substituted ribonucleotide. In one preferred embodiment, the2′-O-alkyl-substituted nucleotide is a 2′-O-methyl ribonucleotide. Inother preferred embodiments, the 3′ and 5′ flanking ribonucleotideregions of an oligonucleotide of the invention comprises at least four2′-O-methyl ribonucleotides. In preferred embodiments, theribonucleotides and deoxyribonucleotides of the hybrid oligonucleotideare linked by phosphorothioate internucleotide linkages. In particularembodiments, this phosphorothioate region or regions have from aboutfour to about 18 nucleosides joined to each other by 5′ to 3′phosphorothioate linkages, and preferably from about 5 to about 18 suchphosphorothioate-linked nucleosides. The phosphorothioate linkages maybe mixed R_(p) and S_(p) enantiomers, or they may be stereoregular orsubstantially stereoregular in either R_(p) or S_(p) form (see Iyer etal. (1995) Tetrahedron Asymmetry 6:1051-1054).

In another preferred embodiment according to this aspect of theinvention, the oligonucleotide is an inverted hybrid oligonucleotidecomprising a region of at least four ribonucleotides flanked by 3′ and5′ deoxyribonucleotide regions of at least two deoxyribonucleotides. Thestructure of this oligonucleotide is “inverted” relative to traditionalhybrid oligonucleotides. In some embodiments, the 2′-O-substituted RNAregion has from about four to about ten 2′-O-substituted nucleosidesjoined to each other by 5′ to 3′ internucleoside linkages, and mostpreferably from about four to about six such 2′-O-substitutednucleosides. In some embodiments, the oligonucleotides of the inventionhave a ribonucleotide region that comprises at least five contiguousribonucleotides. In one particularly preferred embodiment, the overallsize of the inverted hybrid oligonucleotide is 18. In preferredembodiments, the 2′-O-substituted ribonucleosides are linked to eachother through a 5′ to 3′ phosphorothioate, phosphorodithioate,phosphotriester, or phosphodiester linkages. The phosphorothioate 3′ or5′ flanking region (or regions) has from about four to about 18nucleosides joined to each other by 5′ to 3′ phosphorothioate linkages,and preferably from about 5 to about 18 such phosphorothioate-linkednucleosides. In preferred embodiments, the phosphorothioate regions willhave at least 5 phosphorothioate-linked nucleosides. One specificembodiment is an oligonucleotide having substantially the nucleotidesequence set forth in the Sequence Listing as SEQ ID NO:6. In preferredembodiments of this aspect of the invention, the ribonucleotide regioncomprises 2′-O-substituted ribonucleotides, such as 2′-O-alkylsubstituted ribonucleotides. One particularly preferred embodiment is aninverted hybrid oligonucleotide whose ribonucleotide region comprises atleast one 2′-O-methyl ribonucleotide.

In some embodiments, all of the nucleotides in the inverted hybridoligonucleotide are linked by phosphorothioate internucleotide linkages.In particular embodiments, the deoxyribonucleotide flanking region orregions has from about four to about 18 nucleosides joined to each otherby 5′ to 3′ phosphorothioate linkages, and preferably from about 5 toabout 18 such phosphorothioate-linked nucleosides. In some embodiments,the deoxyribonucleotide 3′ and 5′ flanking regions of the invertedhybrid oligonucleotides of the invention have about 5phosphorothioate-linked nucleosides. The phosphorothioate linkages maybe mixed R_(p) and S_(p) enantiomers, or they may be stereoregular orsubstantially stereoregular in either R_(p) or S_(p) form (see Iyer etal. (1995) Tetrahedron Asymmetry 6:1051-1054)

Another embodiment is a composition of matter for inhibiting theexpression of protein kinase A subunit RI_(α) with reduced side effects,the composition comprising an inverted hybrid oligonucleotide accordingto the invention.

Yet another preferred embodiment according to this aspect of theinvention is an inverted chimeric oligonucleotide comprising anoligonucleotide nonionic region of at least four nucleotides flanked byone or more, and preferably two oligonucleotide phosphorothioateregions. Such a chimeric oligonucleotide has a structure that is“inverted” relative to traditional chimeric oligonucleotides. In oneparticular embodiment, an inverted chimeric oligonucleotide of theinvention has substantially the nucleotide sequence set forth in theSequence Listing as SEQ ID NO:1. In preferred embodiments, theoligonucleotide nonionic region comprises about four to about 12nucleotides joined to each other by 5′ to 3′ nonionic linkages. In someembodiments, the nonionic region contains alkylphosphonate and/orphosphoramidate and/or phosphotriester internucleoside linkages. In oneparticular embodiment, the oligonucleotide nonionic region comprises sixnucleotides. In some preferred embodiments, the oligonucleotide has anonionic region having from about six to about eightmethylphosphonate-linked nucleosides, flanked on either side byphosphorothioate regions, each having from about six to about tenphosphorothioate-linked nucleosides. In preferred embodiments, theflanking region or regions are phosphorothioate nucleotides. In someembodiments, the flanking region or regions have from about four toabout 24 nucleosides joined to each other by 5′ to 3′ phosphorothioatelinkages, and preferably from about six to about 16 suchphosphorothioate-linked nucleosides. In preferred embodiments, thephosphorothioate regions have from about five to about 15phosphorothioate-linked nucleosides. The phosphorothioate linkages maybe mixed R_(p) and S_(p) enantiomers, or they may be stereoregular orsubstantially stereoregular in either R_(p) or S_(p) form (see Iyer etal. (1995) Tetrahedron Asymmetry 6:1051-1054).

Another embodiment of this aspect of the invention is a composition ofmatter for inhibiting the expression of protein kinase A subunit RI_(α)with reduced side effects, the composition comprising an invertedchimeric oligonucleotide according to the invention.

Another aspect of the invention is a method of inhibiting theproliferation of cancer cells in vitro. In this method, anoligonucleotide of the invention is administered to the cells.

Yet another aspect is a therapeutic composition comprising anoligonucleotide of the invention in a pharmaceutically acceptablecarrier.

A method of treating cancer in an afflicted subject with reduced sideeffects is another aspect of the invention. This method comprisesadministering a therapeutic composition of the invention to the subjectin which the protein kinase A subunit RI_(α) gene is beingover-expressed.

Those skilled in the art will recognize that the elements of thesepreferred embodiments can be combined and the inventor does contemplatesuch combination. For example, 2′-O-substituted ribonucleotide regionsmay well include from one to all nonionic internucleoside linkages.Alternatively, nonionic regions may have from one to all2′-O-substituted ribonucleotides. Moreover, oligonucleotides accordingto the invention may contain combinations of one or more2′-O-substituted ribonucleotide region and one or more nonionic region,either or both being flanked by phosphorothioate regions. (SeeNucleosides & Nucleotides 14:1031-1035 (1995) for relevant synthetictechniques.)

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects of the present invention, the variousfeatures thereof, as well as the invention itself may be more fullyunderstood from the following description, when read together with theaccompanying drawings in which:

FIG. 1 is a graphic representation showing the effect of modifiedoligonucleotides of the invention on tumor size in a mouse relative tovarious controls.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. The issuedU.S. patents, allowed applications, published foreign applications, andreferences cited herein are hereby incorporated by reference.

Synthetic oligonucleotides of the hybrid, inverted hybrid, and invertedchimeric oligonucleotides as described above.

Such synthetic hybrid, inverted hybrid, and inverted chimericoligonucleotides of the invention have a nucleotide sequencecomplementary to a genomic region or an RNA molecule transcribedtherefrom encoding the RI_(α) subunit of PKA. These oligonucleotides areabout 15 to about 30 nucleotides in length, preferably about 15 to 25nucleotides in length, but most preferably, are about 18 nucleotideslong. The sequence of this gene is known. Thus, an oligonucleotide ofthe invention can have any nucleotide sequence complementary to anyregion of the gene. Three non-limiting examples of an 18mer of theinvention has the sequence set forth below in TABLE 1 as SEQ ID NOS:1,4, and 6.

TABLE 1 Oligo # Sequence (5′ → 3′) Type SEQ ID NO: 164 GCG TGC CTC CTCACT GGC Antisense 1 167 GCG CGC CTC CTC GCT GGC Mismatched 2 Control 188GCA TGC TTC CAC ACA GGC Mismatched 3 Control *** *             * *** 165GCG UGC CTC CTC ACU GGC Hybrid 4 *** *             * *** Mismatched 168GCG CGC CTC CTC GCU GGC Hybrid (Control) 5         *** ** 166 GCG TGCCUC CUC ACT GGC Inverted Hybrid 6         *** ** Mismatched 169 GCG CGCCUC CUC GCT GGC Inverted Hybrid 7 (Control)         *** ** Mismatched189 GCA TGC AUC CGC ACA GGC Inverted Hybrid 8 (Control)         •••  •••190 GCG TGC CTC CTC ACT GGC Inverted Chimeric 1         •••  •••Mismatched 191 GCG CGC CTC CTC GCT GGC Inverted Chimeric 2 (Control) X =mismatched bases * ribonucleotide • methylphosphonate nucleotide

Oligonucleotides having greater than 18 oligonucleotides are alsocontemplated by the invention. These oligonucleotides have up to 25additional nucleotides extending from the 3′, or 5′ terminus, or fromboth the 3′ and 5′ termini of, for example, the 18mer with SEQ ID NOS:1,4, or 6, without diminishing the ability of these oligonucleotides todown regulate RI_(α) gene expression. Alternatively, otheroligonucleotides of the invention may have fewer nucleotides than, forexample, oligonucleotides having SEQ ID NOS:1, 4, or 6. Such shortenedoligonucleotides maintain at least the antisense activity of the parentoligonucleotide to down-regulate the expression of the RI_(α) gene, orhave greater activity.

The oligonucleotides of the invention can be prepared by art recognizedmethods. Oligonucleotides with phosphorothioate linkages can be preparedmanually or by an automated synthesizer and then processed using methodswell known in the field such as phosphoramidite (reviewed in Agrawal etal. (1992) Trends Biotechnol. 10:152-158, see, e.g., Agrawal et al.(1988) Proc. Natl. Acad. Sci. (USA) 85:7079-7083) or H-phosphonate (see,e.g., Froehler (1986) Tetrahedron Lett. 27:5575-5578) chemistry. Thesynthetic methods described in Bergot et al. (J. Chromatog. (1992)559:35-42) can also be used. Examples of other chemical groups includealkylphosphonates, phosphorodithioates, alkylphosphonothioates,phosphoramidates, 2′-O-methyls, carbamates, acetamidate, carboxymethylesters, carbonates, and phosphate triesters. Oligonucleotides with theselinkages can be prepared according to known methods (see, e.g.,Goodchild (1990) Bioconjugate Chem. 2:165-187; Agrawal et al. (Proc.Natl. Acad. Sci. (USA) (1988) 85:7079-7083); Uhlmann et al. (Chem. Rev.(1990) 90:534-583; and Agrawal et al. (Trends Biotechnol. (1992)10:152-158)).

Preferred hybrid, inverted hybrid, and inverted chimericoligonucleotides of the invention may have other modifications which donot substantially affect their ability to specifically down-regulateRI_(α) gene expression. These modifications include those which areinternal or are at the end(s) of the oligonucleotide molecule andinclude additions to the molecule at the internucleoside phosphatelinkages, such as cholesteryl or diamine compounds with varying numbersof carbon residues between the two amino groups, and terminal ribose,deoxyribose and phosphate modifications which cleave, or crosslink tothe opposite chains or to associated enzymes or other proteins whichbind to the RI_(α) nucleic acid. Examples of such oligonucleotidesinclude those with a modified base and/or sugar such as arabinoseinstead of ribose, or a 3′,5′-substituted oligonucleotide having a sugarwhich, at one or both its 3′ and 5′ positions is attached to a chemicalgroup other than a hydroxyl or phosphate group (at its 3′ or 5′position). Other modified oligonucleotides are capped with a nucleaseresistance-conferring bulky substituent at their 3′ and/or 5′ end(s), orhave a substitution in one or both nonbridging oxygens per nucleotide.Such modifications can be at some or all of the internucleosidelinkages, as well as at either or both ends of the oligonucleotideand/or in the interior of the molecule (reviewed in Agrawal et al.(1992) Trends Biotechnol. 10:152-158).

The invention also provides therapeutic compositions suitable fortreating undesirable, uncontrolled cell proliferation or cancercomprising at least one oligonucleotide in accordance with theinvention, capable of specifically down-regulating expression of theRI_(α) gene, and a pharmaceutically acceptable carrier or diluent. It ispreferred that an oligonucleotide used in the therapeutic composition ofthe invention be complementary to at least a portion of the RI_(α)genomic region, gene, or RNA transcript thereof.

As used herein, a “pharmaceutically or physiologically acceptablecarrier” includes any and all solvents (including but not limited tolactose), dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions of the invention is contemplated. Supplementary activeingredients can also be incorporated into the compositions.

Several preferred therapeutic compositions of the invention suitable forinhibiting cell proliferation in vitro or in vivo or for treating cancerin humans in accordance with the methods of the invention comprise about25 to 75 mg of a lyophilized oligonucleotide(s) having SEQ ID NOS:1, 4,and/or 6 and 20-75 mg lactose, USP, which is reconstituted with sterilenormal saline to the therapeutically effective dosages described herein.

The invention also provides methods for treating humans suffering fromdisorders or diseases wherein the RI_(α) gene is incorrectly orover-expressed. Such a disorder or disease that could be treated usingthis method includes tumor-forming cancers such as, but not limited to,human colon carcinoma, breast carcinoma, gastric carcinoma, andneuroblastoma. In the method of the invention, a therapeuticallyeffective amount of a composition of the invention is administered tothe human. Such methods of treatment according to the invention, may beadministered in conjunction with other therapeutic agents.

As used herein, the term “therapeutically effective amount” means thetotal amount of each active component of the pharmaceutical formulationor method that is sufficient to show a meaningful subject or patientbenefit, i.e., a reduction in tumor growth or in the expression ofproteins which cause or characterize the cancer. When applied to anindividual active ingredient, administered alone, the term refers tothat ingredient alone. When applied to a combination, the term refers tocombined amounts of the active ingredients that result in thetherapeutic effect, whether administered in combination, serially orsimultaneously.

A “therapeutically effective manner” refers to a route, duration, andfrequency of administration of the pharmaceutical formulation whichultimately results in meaningful patient benefit, as described above. Insome embodiments of the invention, the pharmaceutical formulation isadministered via injection, sublingually, rectally, intradermally,orally, or enterally in bolus, continuous, intermittent, or continuous,followed by intermittent, regimens.

The therapeutically effective amount of synthetic oligonucleotide in thepharmaceutical composition of the present invention will depend upon thenature and severity of the condition being treated, and on the nature ofprior treatments which the patient has undergone. Ultimately, theattending physician will decide the amount of synthetic oligonucleotidewith which to treat each individual patient. Initially, the attendingphysician will administer low doses of the synthetic oligonucleotide andobserve the patient's response. Larger doses of syntheticoligonucleotide may be administered until the optimal therapeutic effectis obtained for the patient, and at that point the dosage is notincreased further. It is contemplated that the dosages of thepharmaceutical compositions administered in the method of the presentinvention should contain about 0.1 to 5.0 mg/kg body weight per day, andpreferably 0.1 to 2.0 mg/kg body weight per day. When administeredsystemically, the therapeutic composition is preferably administered ata sufficient dosage to attain a blood level of oligonucleotide fromabout 0.01 μM to about 10 μM. Preferably, the concentration ofoligonucleotide at the site of aberrant gene expression should be fromabout 0.01 μM to about 10 μM, and most preferably from about 0.05 μM toabout 5 μM. However, for localized administration, much lowerconcentrations than this may be effective, and much higherconcentrations may be tolerated. It may be desirable to administersimultaneously or sequentially a therapeutically effective amount of oneor more of the therapeutic compositions of the invention to anindividual as a single treatment episode.

Administration of pharmaceutical compositions in accordance with theinvention or to practice the method of the present invention can becarried out in a variety of conventional ways, such as by oralingestion, enteral, rectal, or transdermal administration, inhalation,sublingual administration, or cutaneous, subcutaneous, intramuscular,intraocular, intraperitoneal, or intravenous injection, or any otherroute of administration known in the art for administrating therapeuticagents.

When the composition is to be administered orally, sublingually, or byany non-injectable route, the therapeutic formulation will preferablyinclude a physiologically acceptable carrier, such as an inert diluentor an assimilable edible carrier with which the composition isadministered. Suitable formulations that include pharmaceuticallyacceptable excipients for introducing compounds to the bloodstream byother than injection routes can be found in Remington's PharmaceuticalSciences (18th ed.) (Genarro, ed. (1990) Mack Publishing Co., Easton,Pa.). The oligonucleotide and other ingredients may be enclosed in ahard or soft shell gelatin capsule, compressed into tablets, orincorporated directly into the individual's diet. The therapeuticcompositions may be incorporated with excipients and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. When the therapeuticcomposition is administered orally, it may be mixed with other foodforms and pharmaceutically acceptable flavor enhancers. When thetherapeutic composition is administered enterally, they may beintroduced in a solid, semi-solid, suspension, or emulsion form and maybe compounded with any number of well-known, pharmaceutically acceptableadditives. Sustained release oral delivery systems and/or entericcoatings for orally administered dosage forms are also contemplated suchas those described in U.S. Pat. Nos. 4,704,295, 4,556,552, 4,309,404,and 4,309,406.

When a therapeutically effective amount of composition of the inventionis administered by injection, the synthetic oligonucleotide willpreferably be in the form of a pyrogen-free, parenterally-acceptable,aqueous solution. The preparation of such parenterally-acceptablesolutions, having due regard to ph, isotonicity, stability, and thelike, is within the skill in the art. A preferred pharmaceuticalcomposition for injection should contain, in addition to the syntheticoligonucleotide, an isotonic vehicle such as Sodium Chloride Injection,Ringer's Injection, Dextrose Injection, Dextrose and Sodium ChlorideInjection, Lactated Ringer's Injection, or other vehicle as known in theart. The pharmaceutical composition of the present invention may alsocontain stabilizers, preservatives, buffers, antioxidants, or otheradditives known to those of skill in the art.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile. It must be stableunder the conditions of manufacture and storage and may be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi. The carrier can be a solvent or dispersion medium. The preventionof the action of microorganisms can be brought about by variousantibacterial and antifungal agents. Prolonged absorption of theinjectable therapeutic agents can be brought about by the use ofcompositions of agents delaying absorption. Sterile injectable solutionsare prepared by incorporating the oligonucleotide in the required amountin the appropriate solvent, followed by filtered sterilization.

The pharmaceutical formulation can be administered in bolus, continuous,or intermittent dosages, or in a combination of continuous andintermittent dosages, as determined by the physician and the degreeand/or stage of illness of the patient. The duration of therapy usingthe pharmaceutical composition of the present invention will vary,depending on the unique characteristics of the oligonucleotide and theparticular therapeutic effect to be achieved, the limitations inherentin the art of preparing such a therapeutic formulation for the treatmentof humans, the severity of the disease being treated and the conditionand potential idiosyncratic response of each individual patient.Ultimately the attending physician will decide on the appropriateduration of intravenous therapy using the pharmaceutical composition ofthe present invention.

Compositions of the invention are useful for inhibiting or reducing theproliferation of cancer or tumor cells in vitro. A syntheticoligonucleotide of the invention is administered to the cells in anamount sufficient to enable the binding of the oligonucleotide to acomplementary genomic region or RNA molecule transcribed therefromencoding the RI_(α) subunit. In this way, expression of PKA isdecreased, thus inhibiting or reducing cell proliferation.

Compositions of the invention are also useful for treating cancer oruncontrolled cell proliferation in humans. In this method, a therapeuticformulation including an antisense oligonucleotide of the invention isprovided in a physiologically acceptable carrier. The individual is thentreated with the therapeutic formulation in an amount sufficient toenable the binding of the oligonucleotide to the PKA RI_(α) genomicregion or RNA molecule transcribed therefrom in the infected cells. Inthis way, the binding of the oligonucleotide inhibits or down-regulatesRI_(α) expression and hence the activity of PKA.

In practicing the method of treatment or use of the present invention, atherapeutically effective amount of at least one or more therapeuticcompositions of the invention is administered to a subject afflictedwith a cancer. An anticancer response showing a decrease in tumor growthor size or a decrease in RI_(α) expression is considered to be apositive indication of the ability of the method and pharmaceuticalformulation to inhibit or reduce cell growth and thus, to treat cancerin humans.

At least one therapeutic composition of the invention may beadministered in accordance with the method of the invention either aloneor in combination with other known therapies for cancer such ascisplatin, carboplatin, paclitaxel, tamoxifen, taxol, interferon α anddoxorubicin. When co-administered with one or more other therapies, thecompositions of the invention may be administered either simultaneouslywith the other treatment(s), or sequentially. If administeredsequentially, the attending physician will decide on the appropriatesequence of administering the compositions of the invention incombination with the other therapy.

The following examples illustrate the preferred modes of making andpracticing the present invention, but are not meant to limit the scopeof the invention since alternative methods may be utilized to obtainsimilar results.

EXAMPLE 1 Synthesis, Deprotection, and Purification of Oligonucleotides

Oligonucleotides were synthesized using standard phosphoramiditechemistry (Beaucage (1993) Meth. Mol. Biol. 20:33-61) on an automatedDNA synthesizer (Model 8700, Biosearch, Bedford, Mass.) using abeta-cyanoethyl phosphoramidate approach.

Oligonucleotide phosphorothioates were synthesized using an automatedDNA synthesizer (Model 8700, Biosearch, Bedford, Mass.) using abeta-cyanoethyl phosphoramidate approach on a 10 micromole scale. Togenerate the phosphorothioate linkages, the intermediate phosphitelinkage obtained after each coupling was oxidized using 3H,1,2-benzodithiole-3H-one-1,1-dioxide (see Beaucage, in Protocols forOligonucleotides and Analogs: Synthesis and Properties, Agrawal (ed.),(1993) Humana Press, Totowa, N.J., pp. 33-62). Similar synthesis wascarried out to generate phosphodiester linkages, except that a standardoxidation was carried out using standard iodine reagent. Synthesis ofinverted chimeric oligonucleotide was carried out in the same manner,except that methylphosphonate linkages were assembled using nucleosidemethylphosphonamidite (Glen Research, Sterling, Va.), followed byoxidation with 0.1 M iodine in tetrahydrofuran/2,6-lutidine/water(75:25:0.25) (see Agrawal & Goodchild (1987) Tet. Lett. 28:3539-3542).Hybrids and inverted hybrid oligonucleotides were synthesized similarly,except that the segment containing 2′-O-methylribonucleotides wasassembled using 2′-O-methylribonucleoside phosphoramidite, followed byoxidation to a phosphorothioate or phosphodiester linkage as describedabove. Deprotection and purification of oligonucleotides was carried outaccording to standard procedures, (see Padmapriya et al. (1994)Antisense Res. & Dev. 4:185-199), except for oligonucleotides containingmethylphosphonate-containing regions. For those oligonucleotides, theCPG-bound oligonucleotide was treated with concentrated ammoniumhydroxide for 1 hour at room temperature, and the supernatant wasremoved and evaporated to obtain a pale yellow residue, which was thentreated with a mixture of ethylenediamine/ethanol (1:1 v/v) for 6 hoursat room temperature and dried again under reduced pressure.

EXAMPLE 2 In Vitro Complement Activation Studies

The cell line utilized was the CEM-SS cell line (Southern ResearchInstitute-Frederick Research Center, Frederick, Md.). These cells arehighly susceptible to infection with HIV, rapidly form multinucleatedsyncytia, and are eventually killed by HIV. The cells were maintained(2−7×10⁵ cells per ml) in RPMI 1640 tissue culture medium supplementdwith 10% fetal bovine serum, glutamine, and antibiotics, and werepassaged twice weekly at 1:20 dilution. Passage number was logged eachweek. Cells were discarded after twenty weeks of passage and freshCEM-SS cells thawed and utilized in the assay. Stocks of CEM-SS cellswere frozen in liquid nitrogen in 1 ml NUNC vials in 90% fetal calfserum and 10% dimethyl sulfoxide (DMSO). Following thawing, CEM-SS cellswere routinely ready to be utilized in the primary screen assay aftertwo weeks in culture. Prior to replacing a late passage cell line, thenew CEM-SS cells were tested in the screening assay protocol utilizingthe current stock of infectious virus and AZT. If the infectivity of thevirus was significantly different on the new cells or if AZT appearedless active than expected the new cells were not entered into thescreening program. Mycoplasma testing was routinely performed on allcell lines.

Titer determinations included reverse transcriptase activity assay (seemethods below), endpoint titration or plaque assay (CEM-SS)quantification of infectious particles (see methods below), andquantification of cell killing kinetics.

ELISA kits were purchased from Coulter. The assay is performed accordingto the manufacturer's recommendations. Prior to ELISA analysis weroutinely performed the reverse transcriptase activity assays (describedabove) and used the values for incorporated radioactivity in the RTactivity assay to determine the dilution of our samples required for theELISA. We have constructed standard curves so that the dilutions ofvirus to be used in the p24 ELISA can be accurately determined from theRT activity assay. Control curves are generated in each assay toaccurately quantify the amount of capsid protein in each sample. Datawas obtained by spectrophotometric analysis at 450 nm using a MolecularDevices Vmax plate reader. P24 concentrations were calculated from theoptical density values by use of the Molecular Devices software packageSoft Max.

Infectious virus particles were quantified utilizing the CEM-SS plaqueassay as described by Nara, P. L. and Fischinger, P. J. (1988)Quantitative infectivity assay for HIV-1 and HIV-2 Nature 332:469-470).Flat bottom 96-well microtiter plates (Costar) were coated with 50 μl ofpoly-L-lysine (Sigma) at 50 μg/ml for 2 hours at 37° C. The wells werethen washed with PBS and 2.5×10⁵ CEM-SS cells were placed in themicrotiter well where they became fixed to the bottom of the plate.Enough cells were added to form a monolayer of CEM-SS cells in eachwell. Virus containing supernatant was added from each well of the XTTplate, including virus and cell controls and each serial dilution of thetest substance. The number of syncytia were quantified in theflat-bottom 96-well microtiter plate with an Olympus CK2 invertedmicroscope at 4 days following infection. Each syncytium resulted from asingle infectious HIV virion.

To determine the relative effect of inverted hybrid or inverted chimericstructure on oligonucleotide-mediated depletion of complement, thefollowing experiments were performed. Venous blood was collected fromhealthy adult human volunteers. Serum was prepared for hemolyticcomplement assay by collecting blood into vacutainers (Becton Dickinson#6430 Franklin Lakes, N.J.) without commercial additives. Blood wasallowed to clot at room temperature for 30 minutes, chilled on ice for15 minutes, then centrifuged at 4° C. to separate serum. Harvested serumwas kept on ice for same day assay or, alternatively, stored at −70° C.Buffer, or an oligonucleotide sample was then incubated with the serum.The oligonucleotides tested were 25mer oligonucleotide phosphodiestersor phosphorothioates, 25mer hybrid oligonucleotides, 25mer invertedhybrid oligonucleotides, 25mer chimeric oligonucleotides, and 25merinverted chimeric oligonucleotides. Representative hybridoligonucleotides were composed of seven to 13 2′-O-methylribonucleotides flanked by two regions of six to ninedeoxyribonucleotides each. Representative 25mer inverted hybridoligonucleotides were composed of 17 deoxyribonucleotides flanked by tworegions of four ribonucleotides each. Representative 25mer chimericoligonucleotides were composed of six methylphosphonatedeoxyribonucleotides and 19 phosphorothioate deoxyribonucleotides.Representative inverted chimeric oligonucleotides were composed of from16 to 17 phosphorothioate deoxyribonucleotides flanked by regions offrom two to seven methylphosphonate deoxyribonucleotides, or from six toeight methylphosphonate deoxyribonucleotides flanked by nine to tenphosphorothioate deoxyribonucleotides, or two phosphorothioate regionsranging from two to 12 oligonucleotides, flanked by threephosphorothioate regions ranging in size from two to six nucleotides inlength. A standard CH50 assay (See Kabat and Mayer (eds), ExperimentalImmunochemistry, 2d Ed., Springfield, Ill., C. C. Thomas, p. 125) forcomplement-mediated lysis of sheep red blood cells (Colorado Serum Co.)sensitized with anti-sheep red blood cell antibody (hemolysin, Diamedix,Miami, Fla.) was performed, using duplicate determinations of at leastfive dilutions of each test serum, then hemoglobin release intocell-free supernates was measured spectrophotometrically at 541 nm.

EXAMPLE 3 In Vitro Mitogenicity Studies

To determine the relative effect of inverted hybrid or inverted chimericstructure on oligonucleotide-mediated mitogenicity, the followingexperiments were performed. Spleen was taken from a male CD1 mouse (4-5weeks, 20-22 g; Charles River, Wilmington, Mass.). Single cellsuspensions were prepared by gently mincing with frosted edges of glassslides. Cells were then cultured in RPMI complete media (RPMI mediasupplemented with 10% fetal bovine serum (FBS), 50 micromolar2-mercaptoethanol (2-ME), 100 U/ml penicillin, 100 micrograms/mlstreptomycin, 2 mM L-glutamine). To minimize oligonucleotidedegradation, FBS was first heated for 30 minutes at 65° C.(phosphodiester-containing oligonucleotides) or 56° C. (all otheroligonucleotides). Cells were plated in 96 well dishes at 100,000 cellsper well (volume of 100 microliters/well). One type of eacholigonucleotide described in Example 2 above in 10 microliters TE buffer(10 mM Tris-HCl, pH 7.5, 1 mM EDTA) was added to each well. After 44hours of culturing at 37° C., one microcurie tritiated thymidine(Amersham, Arlington Heights, Ill.) was added in 20 microliters RPMImedia for a 4 hour pulse labelling. The cells were then harvested in anautomatic cell harvester (Skatron, Sterling, Va.) and the filters wereassessed using a scintillation counter. In control experiments formitogenicity, cells were treated identically, except that either media(negative control) or concanavalin A (positive control) was added to thecells in place of the oligonucleotides.

All of the inverted hybrid oligonucleotides proved to be lessimmunogenic than phosphorothioate oligonucleotides. Inverted hybridoligonucleotides having phosphodiester linkages in the 2′-O-methylregion appeared to be slightly less immunogenic than those containingphosphorothioate linkages in that region. No significant difference inmitogenicity was observed when the 2′-O-methyl ribonucleotide region waspared down from 13 to 11 or to 9 nucleotides. Inverted chimericoligonucleotides were also generally less mitogenic thanphosphorothioate oligonucleotides. In addition, these oligonucleotidesappeared to be less mitogenic than traditional chimericoligonucleotides, at least in cases in which the traditional chimericoligonucleotides had significant numbers of methylphosphonate linkagesnear the 3′ end. Increasing the number of methylphosphonate linkers inthe middle of the oligonucleotide from 5 to 6 or 7 did not appear tohave a significant effect on mitogenicity. These results indicate thatincorporation of inverted hybrid or inverted chimeric structure into anoligonucleotide can reduce its mitogenicity.

EXAMPLE 4 In Vitro Studies

To determine the relative effect of inverted hybrid or inverted chimericstructure on oligonucleotide-induced mitogenicity, the followingexperiments were performed. Venous blood was collected from healthyadult human volunteers. Plasma for clotting time assay was prepared bycollecting blood into siliconized vacutainers with sodium citrate(Becton Dickinson #367705), followed by two centrifugations at 4° C. toprepare platelet-poor plasma. Plasma aliquots were kept on ice, spikedwith various test oligonucleotides described in Example 2 above, andeither tested immediately or quickly frozen on dry ice for subsequentstorage at −20° C. prior to coagulation assay. Activated partialthromboplastin time (aPTT) was performed in duplicate on an Electra1000C (Medical Laboratory Automation, Mount Vernon, N.Y.) according tothe manufacturer's recommended procedures, using Actin FSL (Baxter Dade,Miami, Fla.) and calcium to initiate clot formation, which was measuredphotometrically. Prolongation of aPTT was taken as an indication ofclotting inhibition side effect produced by the oligonucleotide.

Traditional phosphorothioate oligonucleotides produced the greatestprolongation of aPTT, of all of the oligonucleotides tested. Traditionalhybrid oligonucleotides produced somewhat reduced prolongation of aPTT.In comparison with traditional phosphorothioate or traditional hybridoligonucleotides, all of the inverted hybrid oligonucleotides testedproduced significantly reduced prolongation of aPTT. Inverted hybridoligonucleotides having phosphodiester linkages in the 2′-O-substitutedribonucleotide region had the greatest reduction in this side effect,with one such oligonucleotide having a 2′-O-methyl RNA phosphodiesterregion of 13 nucleotides showing very little prolongation of aPTT, evenat oligonucleotide concentrations as high as 100 micrograms/ml.Traditional chimeric oligonucleotides produce much less prolongation ofaPTT than do traditional phosphorothioate oligonucleotides. Generally,inverted chimeric oligonucleotides retain this characteristic. At leastone inverted chimeric oligonucleotide, having a methylphosphonate regionof seven nucleotides flanked by phosphorothioate regions of ninenucleotides, gave better results in this assay than the traditionalchimeric oligonucleotides at all but the highest oligonucleotideconcentrations tested. These results indicate that inverted hybrid andinverted chimeric oligonucleotides may provide advantages in reducingthe side effect of clotting inhibition when they are administered tomodulate gene expression in vivo.

EXAMPLE 5 In Vivo Complement Activation Studies

Rhesus monkeys (4-9 kg body weight) are acclimatized to laboratoryconditions for at least 7 days prior to the study. On the day of thestudy, each animal is lightly sedated with ketamine-HCl (10 mg/kg) anddiazepam (0.5 mg/kg). Surgical level anesthesia is induced andmaintained by continuous ketamine intravenous drip throughout theprocedure. The oligonucleotides described in Example 2 above aredissolved in normal saline and infused intravenously via a cephalic veincatheter, using a programmable infusion pump at a delivery rate of 0.42mg/minute. For each oligonucleotide, doses of 0, 0.5, 1, 2, 5 and 10mg/kg are administered to two animals each over a 10 minute infusionperiod. Arterial blood samples are collected 10 minutes prior tooligonucleotide administration and 2, 5, 10, 20, 40 and 60 minutes afterthe start of the infusion, as well as 24 hours later. Serum is used fordetermining complement CH50, using the conventional complement-dependentlysis of sheep erythrocyte procedure (see Kabat and Mayer, 1961, supra).At the highest dose, phosphorothioate oligonucleotide causes a decreasein serum complement CH50 beginning within 5 minutes of the start ofinfusion. Inverted hybrid and chimeric oligonucleotides are expected toshow a much reduced or undetectable decrease in serum complement CH50under these conditions.

EXAMPLE 6 In Vivo Mitogenicity Studies

CD1 mice are injected intraperitoneally with a dose of 50 mg/kg bodyweight of oligonucleotide described in Example 2 above. Forty-eighthours later, the animals are euthanized and the spleens are removed andweighed. Animals treated with inverted hybrid or inverted hybridoligonucleotides are expected to show no significant increase in spleenweight, while those treated with oligonucleotide phosphorothioates areexpected to show modest increases in spleen weight.

EXAMPLE 7 In Vivo Clotting Studies

Rhesus monkeys are treated as in Example 5. From the whole blood samplestaken, plasma for clotting assay is prepared, and the assay performed,as described in Example 4. It is expected that prolongation of aPTT willbe substantially reduced for both inverted hybrid oligonucleotides andfor inverted chimeric oligonucleotide, relative to traditionaloligonucleotide phosphorothioates.

EXAMPLE 8 RNase H Activity Studies

To determine the ability of inverted hybrid oligonucleotides andinverted chimeric oligonucleotides to activate RNase H when bound to acomplementary RNA molecule, the following experiments were performed.Each type of oligonucleotide described in Example 2 above was incubatedtogether with a molar equivalent quantity of complimentaryoligoribonucleotide (0.266 micromolar concentration of each), in acuvette containing a final volume of 1 ml RNase H buffer (20 mMTris-HCl, pH 7.5, 10 mM MgCl₂, 0.1 M KCl, 2% glycerol, 0.1 mM DTT). Thesamples were heated to 95° C., then cooled gradually to room temperatureto allow annealing to form duplexes. Annealed duplexes were incubatedfor 10 minutes at 37° C., then 5 units RNase H was added and datacollection commenced over a three hour period. Data was collected usinga spectrophotometer (GBC 920, GBC Scientific Equipment, Victoria,Australia) at 259 nm. RNase H degradation was determined by hyperchromicshift.

As expected, phosphodiester oligonucleotides behaved as very goodco-substrates for RNase H-mediated degradation of RNA, with adegradative half-life of 8.8 seconds. Phosphorothioate oligonucleotidesproduced an increased half-life of 22.4 seconds. Introduction of a2′-O-methyl ribonucleotide segment at either end of the oligonucleotidefurther worsened RNase H activity (half-life=32.7 seconds). In contrast,introducing a 2′-O-methyl segment into the middle of the oligonucleotide(inverted hybrid structure) always resulted in improved RNase H-mediateddegradation. When a region of 13 2′-O-methylribonucleosidephosphodiesters was flanked on both sides by phosphorothioate DNA, thebest RNase H activity was observed, with a half-life of 7.9 seconds.Introduction of large blocks of methylphosphonate-linked nucleosides atthe 3′ end of the oligonucleotide either had no effect or caused furtherdeterioration of RNase H activity even when in a chimeric configuration.Introduction of methylphosphonate linked nucleosides at the 5′ end,however, improved RNase H activity, particularly in a chimericconfiguration with a single methylphosphonate linker at the 3′ end (besthalf-life=8.1 seconds). All inverted chimeric oligonucleotides withmethylphosphonate core regions flanked by phosphorothioate regions gavegood RNase results, with a half-life range of 9.3 to 14.4 seconds. Theseresults indicate that the introduction of inverted hybrid or invertedchimeric structure into phosphorothioate-containing oligonucleotides canrestore some or all of the ability of the oligonucleotide to act as aco-substrate for RNase H, a potentially important attribute for aneffective antisense agent.

EXAMPLE 9 Melting Temperature Studies

To determine the effect of inverted hybrid or inverted chimericstructure on stability of the duplex formed between an antisenseoligonucleotide and a target molecule, the following experiments wereperformed. Thermal melting (Tm) data were collected using aspectrophotometer (GBC 920, GBC Scientific Equipment, Victoria,Australia), which has six 10 mm cuvettes mounted in a dual carousel. Inthe Tm experiments, the temperature was directed and controlled througha peltier effect temperature controller by a computer, using softwareprovided by GBC, according to the manufacturer's directions. Tm datawere analyzed by both the first derivative method and the mid-pointmethod, as performed by the software. Tm experiments were performed in abuffer containing 10 mM PIPES, pH 7.0, 1 mM EDTA, 1 M NaCl. Arefrigerated bath (VWR 1166, VWR, Boston, Mass.) was connected to thepeltier-effect temperature controller to absorb the heat oligonucleotidestrand concentration was determined using absorbance values at 260 nm,taking into account extinction coefficients.

EXAMPLE 10 Tumor Growth and Antisense Treatment

LS-174T human colon carcinoma cells (1×10⁶ cells) were inoculatedsubcutaneously (s.c.) into the left flank of athymic mice. A single doseof RI_(α) antisense hybrid (Oligo 165, SEQ ID NO:4), inverted hybrid(Oligo 166, SEQ ID NO:6), or antisense (Oligo 164, SEQ ID NO:1)oligonucleotides or control oligonucleotide (Oligo 169, SEQ ID NO:7);Oligo 168 (SEQ ID NO:5); Oligo 188, (SEQ ID NO:3) as shown in Table 1 (1mg per 0.1 ml saline per mouse), or saline (0.1 ml per mouse), wasinjected s.c. into the right flank of mice when tumor size reached 80 to100 mg, about 1 week after cell inoculation. Tumor volumes were obtainedfrom daily measurement of the longest and shortest diameters andcalculation by the formula, 4/3πr³ where r=(length+width)/4. At eachindicated time, two animals from the control and antisense-treatedgroups were killed, and tumors were removed and weighed. The results areshown in FIG. 1. These results show that the size of the tumor in theanimal treated with the inverted hybrid oligonucleotide 166 having SEQID NO:6 was surprisingly smaller from three days after injection onwardthan the phosphorothioate oligonucleotide 164 having SEQ ID NO:1. Thatthis effect was sequence-specific is also demonstrated in FIG. 1:control oligonucleotide 168 (SEQ ID NO:5) has little ability to keeptumor size at a minimum relative to the hybrid and inverted hybridoligonucleotides.

EXAMPLE 11 Photoaffinity Labelling and Immunoprecipitation of RI_(α)Subunits

The tumors are homogenized with a Teflon/glass homogenizer in ice-coldbuffer 10 (Tris-HCl, pH 7.4, 20 mM; NaCl, 100 mM; NP-40, 1%; sodiumdeoxycholate, 0.5%; MgCl₂, 5 mM; pepstatin, 0.1 mM; antipain, 0.1 mM;chymostatin, 0.1 mM; leupeptin, 0.2 mM; aprotinin, 0.4 mg/ml; andsoybean trypsin inhibitor, 0.5 mg/ml; filtered through a 0.45-μm poresize membrane), and centrifuged for 5 min in an Eppendorf microfuge at4° C. The supernatants are used as tumor extracts.

The amount of PKA RI_(α) subunits in tumors is determined byphotoaffinity labelling with 8-N₃-[³²P] cAMP followed byimmunoprecipitation with RI_(α) antibodies as described by Tortora etal. (Proc. Natl. Acad. Sci. (USA) (1990) 87:705-708). The photoactivatedincorporation of 8-N₃-[³²p] cAMP (60.0 Ci/m-mol), and theimmunoprecipitation using the anti-RI_(α) or anti-RII_(β) antiserum andprotein A Sepharose and SDS-PAGE of solubilized antigen-antibody complexfollows the method previously described (Tortora et al. (1990) Proc.Natl. Acad. Sci. (USA) 87:705-708; Ekanger et al. (1985) J. Biol. Chem.260:3393-3401). It is expected that the amount of RI_(α) in tumorstreated with hybrid, inverted hybrid, and inverted chimericoligonucleotides of the invention will be reduced compared with theamount in tumors treated with mismatch, straight phosphorothioate, orstraight phosphodiester oligonucleotide controls, saline, or othercontrols.

EXAMPLE 12 cAMP-Dependent Protein Kinase Assays

Extracts (10 mg protein) of tumors from antisense-, control antisense-,or saline-treated animals are loaded onto DEAE cellulose columns (1×10cm) and fractionated with a linear salt gradient (Rohlff et al. (1993)J. Biol. Chem. 268:5774-5782). PKA activity is determined in the absenceor presence of 5 μM cAMP as described below (Rohlff et al. (1993) J.Biol. Chem. 268:5774-5782). cAMP-binding activity is measured by themethod described previously and expressed as the specific binding(Tagliaferri et al. (1988) J. Biol. Chem. 263:409-416).

After two washes with Dulbecco's phosphate-buffered saline, cell pellets(2×10⁶ cells) are lysed in 0.5 ml of 20 mM Tris (pH 7.5), 0.1 mM sodiumEDTA, 1 mm dithiothreitol, 0.1 mM pepstatin, 0.1 mM antipain, 0.1 mMchymostatin, 0.2 mM leupeptin, 0.4 mg/ml aprotinin, and 0.5 mg/mlsoybean trypsin inhibitor, using 100 strokes of a Dounce homogenizer.After centrifugation (Eppendorf 5412) for 5 min, the supernatants areadjusted to 0.7 mg protein/ml and assayed (Uhler et al. (1987) J. Biol.Chem. 262:15202-15207) immediately. Assays (40 μl total volume) areperformed for 10 min at 300° C. and contained 200 μM ATP, 2.7×10⁶ cpm γ[³²P]ATP, 20 mM MgCl₂, 100 μM Kemptide (Sigma K-1127) (Kemp et al.(1977) J. Biol. Chem. 252:4888-4894), 40 mM Tris (pH 7.5), ±100 μMprotein kinase inhibitor (Sigma P-3294) (Cheng et al. (1985) Biochem. J.231:655-661), ±8 μM cAMP and 7 μg of cell extract. The phosphorylationof Kemptide is determined by spotting 20 μl of incubation mixture onphosphocellulose filters (Whatman, P81) and washing in phosphoric acidas described (Roskoski (1983) Methods Enzymol. 99:3-6). Radioactivity ismeasured by liquid scintillation using Econofluor-2 (NEN ResearchProducts NEF-969). It is expected that PKA and cAMP binding activitywill be reduced in extracts of tumors treated with the hybrid, invertedhybrid, and inverted chimeric oligonucleotides of the invention.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific substances and procedures described herein. Such equivalentsare considered to be within the scope of this invention, and are coveredby the following claims.

3 1 27 DNA Artificial Sequence Description of Artificial Sequencecomplementary oligonucleotide to the rev gene of HIV-1 1 tcgtcgctgtctccgcttct tcttgcc 27 2 29 DNA Artificial Sequence Description ofArtificial SequenceCpG in HYB-0272 2 tccatgacgt tcctgatgct ttttggggg 293 29 DNA Artificial Sequence Description of Artificial SequenceGpC inHYB-0352 3 tccatgagct tcctgatgct ttttggggg 29

What is claimed is:
 1. A synthetic, modified oligonucleotidecomplementary to, and capable of down-regulating the expression of,nucleic acid encoding protein kinase A subunit RIα, the modifiedoligonucleotide having from about 15 to about 30 nucleotides and being ahybrid, inverted hybrid, or inverted chimeric oligonucleotide, thehybrid oligonucleotide comprising a region of at least twodeoxyribonucleotides, flanked by 3′ and 5′ flanking ribonucleotideregions each having at least four ribonucleotides, the inverted hybridoligonucleotide comprising a region of at least four ribonucleotidesflanked by 3′ and 5′ flanking deoxyribonucleotide regions of at leasttwo deoxyribonucleotides, and the inverted chimeric oligonucleotidecomprising an oligonucleotide nonionic region of at least fournucleotides flanked by two oligonucleotide phosphorothioate regions. 2.The oligonucleotide of claim 1 having 18 nucleotides.
 3. Theoligonucleotide of claim 1 which is a hybrid oligonucleotide.
 4. Theoligonucleotide of claim 3 consisting essentially of the nucleotidesequence set forth in SEQ ID NO:4.
 5. The oligonucleotide of claim 3,wherein each of the flanking ribonucleotide regions comprises at leastfour contiguous 2′-O-substituted ribonucleotides.
 6. The oligonucleotideof claim 5, wherein each of the flanking ribonucleotide regionscomprises at least one 2′-O-alkyl ribonucleotide.
 7. The oligonucleotideof claim 6, wherein each of the flanking ribonucleotide regionscomprises at least one 2′-O-methyl ribonucleotide.
 8. Theoligonucleotide of claim 5, wherein each of the flanking ribonucleotideregions comprises at least four 2′-O-methyl ribonucleotides.
 9. Theoligonucleotide of claim 3, wherein the ribonucleotides anddeoxyribonucleotides are linked by phosphorothioate internucleotidelinkages.
 10. A therapeutic composition comprising the oligonucleotideof claim 3 in a pharmaceutically acceptable carrier.
 11. A compositionfor inhibiting the expression of protein kinase A with reducedmitogenicity, reduced activation of complement, or reducedantithrombotic properties, relative to the side effects caused by anoligonucleotide which is not hybrid, the composition comprising thehybrid oligonucleotide of claim
 3. 12. The oligonucleotide of claim 1which is an inverted hybrid oligonucleotide.
 13. The oligonucleotide ofclaim 12 consisting essentially of the nucleotide sequence set forth inthe Sequence Listing as SEQ ID NO:6.
 14. The oligonucleotide of claim12, wherein the ribonucleotide region comprises at least five contiguousribonucleotides.
 15. The oligonucleotide of claim 14, wherein thedeoxyribonucleotide flanking regions comprise six contiguousribonucleotides.
 16. The oligonucleotide of claim 12, wherein theflanking ribonucleotide regions comprise 2′-O-substitutedribonucleotides.
 17. The oligonucleotide of claim 16, wherein the2′-O-substituted ribonucleotides are 2′-O-alkyl substitutedribonucleotides.
 18. The oligonucleotide of claim 17, wherein each ofthe flanking ribonucleotide regions comprise at least one 2′-O-methylribonucleotide.
 19. The oligonucleotide of claim 12, wherein thenucleotides are linked by phosphorothioate internucleotide linkages. 20.A therapeutic composition comprising the oligonucleotide of claim 12 ina pharmaceutically acceptable carrier.
 21. The oligonucleotide of claim1 which is an inverted chimeric oligonucleotide.
 22. The oligonucleotideof claim 21 consisting essentially of the nucleotide sequence set forthin the Sequence Listing as SEQ ID NO:1.
 23. The oligonucleotide of claim21, wherein the oligonucleotide nonionic region comprises about 4 toabout 12 nucleotides.
 24. The oligonucleotide of claim 23, wherein theoligonucleotide nonionic region comprises six nucleotides.
 25. Theoligonucleotide of claim 21, wherein the oligonucleotide nonionic regioncomprises alkylphosphonate nucleotides.
 26. The oligonucleotide of claim25, wherein the oligonucleotide nonionic region comprisesmethylphosphonate nucleotides.
 27. The oligonucleotide of claim 21,wherein the nucleotides in the flanking regions comprise at least sixcontiguous nucleotides linked by phosphorothioate internucleotidelinkages.
 28. A therapeutic composition comprising the oligonucleotideof claim 21 in a pharmaceutically acceptable carrier.
 29. A therapeuticcomposition comprising the oligonucleotide of claim 1 in apharmaceutically acceptable carrier.
 30. A composition of matter forinhibiting the expression of protein kinase A with reduced mitogenicity,reduced activation of complement, or reduced antithrombotic properties,relative to the side effects caused by an oligonucleotide which is notinverted hybrid, the composition comprising the inverted hybridoligonucleotide of claim
 12. 31. A composition of matter for inhibitingthe expression of the protein kinase A RI_(α) subunit gene with reducedmitogenicity, reduced activation of complement, or reducedantithrombotic properties, relative to the side effects caused by anoligonucleotide which is not an inverted chimeric, the compositioncomprising the inverted chimeric oligonucleotide of claim 21.